Search

The interface between the cellular actin network and diverse forms of integrin-mediated cell adhesions displays a unique capacity to serve as accurate chemical and mechanical sensors of the cell's microenvironment. Focal adhesion-like structures of diverse cell types, podosomes in osteoclasts, and invadopodia of invading cancer cells display distinct morphologies and apparent functions. Yet, all three share a similar composition and mode of coupling between a protrusive structure (the lamellipodium, the core actin bundle of the podosome, and the invadopodia protrusion, respectively), and a nearby adhesion site. Cytoskeletal or external forces, applied to the adhesion sites, trigger a cascade of unfolding and activation of key adhesome components (e.g., talin, vinculin, integrin), which in turn, trigger the assembly of adhesion sites and generation of adhesion-mediated signals that affect cell behavior and fate. The structural and molecular mechanisms underlying the dynamic crosstalk between the actin cytoskeleton and the adhesome network are discussed. [ABSTRACT FROM AUTHOR]

The mitochondrial voltage-dependent anion channel-1 (VDAC1) protein functions in a variety of mitochondria-linked physiological and pathological processes, including metabolism and cell signaling, as well as in mitochondria-mediated apoptosis. VDAC1 interacts with about 150 proteins to regulate the integration of mitochondrial functions with other cellular activities. Recently, we developed VDAC1-based peptides that have multiple effects on cancer cells and tumors including apoptosis induction. Here, we designed several cell-penetrating VDAC1 N-terminal-derived peptides with the goal of identifying the shortest peptide with improved cellular stability and activity. We identified the D-Δ(1-18)N-Ter-Antp comprising the VDAC1 N-terminal region (19–26 amino acids) fused to the Antp, a cell-penetrating peptide. We demonstrated that this peptide induced apoptosis, autophagy, senescence, cell volume enlargement, and the refusion of divided daughter cells into a single cell, it was responsible for reorganization of actin and tubulin filaments, and increased cell adhesion. In addition, the peptide induced alterations in the expression of proteins associated with cell metabolism, signaling, and division, such as enhancing the expression of nuclear factor kappa B and decreasing the expression of the nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha. These cellular effects may result from the peptide interfering with VDAC1 interaction with its interacting proteins, thereby blocking multiple mitochondrial/VDAC1 pathways associated with cell functions. The results of this study further support the role of VDAC1 as a mitochondrial gatekeeper protein in controlling a variety of cell functions via interaction with associated proteins. [ABSTRACT FROM AUTHOR]

The growth of branched actin networks powers cell-edge protrusions and motility. A heterogeneous density of actin, which yields to a tunable cellular response, characterizes these dynamic structures. We study how actin organization controls both the rate and the steering during lamellipodium growth. We use a high-resolution surface structuration assay combined with mathematical modeling to describe the growth of a reconstituted lamellipodium. We demonstrate that local monomer depletion at the site of assembly negatively impacts the network growth rate. At the same time, network architecture tunes the protrusion efficiency, and regulates the rate of growth. One consequence of this interdependence between monomer depletion and network architecture effects is the ability of heterogeneous network to impose steering during motility. Therefore, we have established that the general principle, by which the cell can modulate the rate and the direction of a protrusion, is by varying both density and architecture of its actin network. [ABSTRACT FROM AUTHOR]

Tight coupling between biochemical and mechanical properties of the actin cytoskeleton drives a large range of cellular processes including polarity establishment, morphogenesis, and motility. This is possible because actin filaments are semi-flexible polymers that, in conjunction with the molecular motor myosin, can act as biological active springs or "dashpots" (in laymen's terms, shock absorbers or fluidizers) able to exert or resist against force in a cellular environment. To modulate their mechanical properties, actin filaments can organize into a variety of architectures generating a diversity of cellular organizations including branched or crosslinked networks in the lamellipodium, parallel bundles in filopodia, and antiparallel structures in contractile fibers. In this review we describe the feedback loop between biochemical and mechanical properties of actin organization at the molecular level in vitro, then we integrate this knowledge into our current understanding of cellular actin organization and its physiological roles. [ABSTRACT FROM AUTHOR]

Cells use their dynamic actin network to control their mechanics and motility. These networks are made of branched actin filaments generated by the Arp2/3 complex. Here we study under which conditions the microscopic organization of branched actin networks builds up a sufficient stress to trigger sustained motility. In our experimental setup, dynamic actin networks or "gels" are grown on a hard bead in a controlled minimal protein system containing actin monomers, profilin, the Arp2/3 complex and capping protein. We vary protein concentrations and follow experimentally and through simulations the shape and mechanical properties of the actin gel growing around beads. Actin gel morphology is controlled by elementary steps including "primer" contact, growth of the network, entanglement, mechanical interaction and force production. We show that varying the biochemical orchestration of these steps can lead to the loss of network cohesion and the lack of effective force production. We propose a predictive phase diagram of actin gel fate as a function of protein concentrations. This work unveils how, in growing actin networks, a tight biochemical and physical coupling smoothens initial primer-caused heterogeneities and governs force buildup and cell motility. [ABSTRACT FROM AUTHOR]

Gazing at a giant redwood tree in the Pacific Northwest, that has grown to enormous heights over centuries, does little to convince one that plants are built for speed and versatility. Even at the cellular level, a system of polymers—the cell skeleton or cytoskeleton—integrates signals and generates subcellular structures spanning scales of a few nanometers to hundreds of micrometers that coordinate cell growth. The term cytoskeleton itself connotes a stable structure. Clearly, this is not the case. Recent studies using advanced imaging modalities reveal the plant actin cytoskeleton to be a highly dynamic, ever changing assemblage of polymers. These insights along with growing evidence about the biochemical/biophysical properties of plant cytoskeletal polymers, especially those obtained by single filament imaging and reconstituted systems of purified proteins analyzed by total internal reflection fluorescence microscopy, allow the generation of a unique model for the dynamic turnover of actin filaments, termed stochastic dynamics. Here, we review several significant advances and highlight opportunities that will position plants at the vanguard of research on actin organization and turnover. A challenge for the future will be to apply the power of reverse-genetics in several model organisms to test the molecular details of this new model. [ABSTRACT FROM AUTHOR]

Actin filaments constitute one of the main components of cell cytoskeleton. Assembled into bundles in filopodia or in stress fibres, they play a pivotal role in eukaryotes during cell morphogenesis, adhesion and motility. The bundle emergence has been extensively related to specific actin regulators in vivo. Such dynamic modulation was also highlighted by biochemical reconstitution of the actin-network assembly, in bulk solution or with biomimetic devices. However, the question of how geometrical boundaries, such as those encountered in cells, affect the dynamic formation of highly ordered actin structures remains poorly studied. Here we demonstrate that the nucleation geometry in itself can be the principal determinant of actin-network architecture. We developed a micropatterning method that enables the spatial control of actin nucleation sites for in vitro assays. Shape, orientation and distance between nucleation regions control filament orientation and length, filament-filament interactions and filopodium-like bundle formation. Modelling of filament growth and interactions demonstrates that basic mechanical and probabilistic laws govern actin assembly in higher-order structures. [ABSTRACT FROM AUTHOR]

Examines the effect of protein VASP on the filament branching/debranching activity of ActA-Arp2/3 complex in relation with its function in motility of Listeria and ActA-coated beads. Cellular factors responsible for the stimulation of actin polymerization at the bacterial surface; Association between ATP hydrolysis and actin polymerization.

In this issue of Chemistry and Biology, Rizvi and colleagues indentify a small molecule that inhibits formin-mediated actin assembly. Together with recently characterized inhibitors of the Arp2/3 complex () and formins (), these small molecules provide useful laboratory tools to dissect the link between actin nucleators and actin-based structures in living cells. [Copyright &y& Elsevier]

Summary: Cells use actin assembly to generate forces for membrane protrusions during movement or, in the case of pathogens, to propel themselves in the host cells, in crude extracts , or in mixtures of actin and other purified proteins . Significant progress has been made in understanding the mechanism of actin-based motility at a macroscopic level by using biomimetic systems in vitro . Here, we combined such a system with evanescent wave microscopy to visualize Arp2/3-mediated actin network formation at single-actin-filament resolution. We found that actin filaments that we call “primers” determine the origin of the autocatalytic and propagative formation of the actin network. In the presence of capping protein, multiple “primers” generate independent networks that merge around the object to form an outer “shell” made of entangled and capped filaments. Simultaneously, newly created filaments on the surface of the particle initiate mechanical stress, which develops until symmetry breaking. Our results and extensive modeling support that the stress, which releases into propulsive forces , is controlled not by any specific orientation of actin filaments toward the nucleation sites but only by new monomers added near the load surface. [Copyright &y& Elsevier]

Summary: Background: The rapid dynamics of actin filaments is a fundamental process that powers a large number of cellular functions. However, the basic mechanisms that control and coordinate such dynamics remain a central question in cell biology. To reach beyond simply defining the inventory of molecules that control actin dynamics and to understand how these proteins act synergistically to modulate filament turnover, we combined evanescent-wave microscopy with a biomimetic system and followed the behavior of single actin filaments in the presence of a physiologically relevant mixture of accessory proteins. This approach allows for the real-time visualization of actin polymerization and age-dependent filament severing. Results: In the presence of actin-depolymerizing factor (ADF)/cofilin and profilin, actin filaments with a processive formin attached at their barbed ends were observed to oscillate between stochastic growth and shrinkage phases. Fragmentation of continuously growing actin filaments by ADF/cofilin is the key mechanism modulating the prominent and frequent shortening events. The net effect of continuous actin polymerization, driven by a processive formin that uses profilin-actin, and of ADF/cofilin-mediating severing that trims the aged ends of the growing filaments is an up to 155-fold increase in the rate of actin-filament turnover in vitro in comparison to that of actin alone. Lateral contact between actin filaments dampens the dynamics and favors actin-cable formation. A kinetic simulation accurately validates these observations. Conclusions: Our proposed mechanism for the control of actin dynamics is dominated by ADF/cofilin-mediated filament severing that induces a stochastic behavior upon individual actin filaments. When combined with a selection process that stabilizes filaments in bundles, this mechanism could account for the emergence and extension of actin-based structures in cells. [Copyright &y& Elsevier]

Bundles of actin filaments are central to a large variety of cellular structures such as filopodia, stress fibers, cytokinetic rings, and focal adhesions. The mechanical properties of these bundles are critical for proper force transmission and force bearing. Previous mathematical modeling efforts have focused on bundles' rigidity and shape. However, it remains unknown how bundle length and buckling are controlled by external physical factors. In this work, we present a biophysical model for dynamic bundles of actin filaments submitted to an external load. In combination with in vitro motility assays of beads coated with formins, our model allowed us to characterize conditions for bead movement and bundle buckling. From the deformation profiles, we determined key biophysical properties of tethered actin bundles such as their rigidity and filament density., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Principles of regulation of actin network dimensions are fundamentally important for cell functions, yet remain unclear. Using both in vitro and in silico approaches, we studied the effect of key parameters, such as actin density, ADF/Cofilin concentration and network width on the network length. In the presence of ADF/Cofilin, networks reached equilibrium and became treadmilling. At the trailing edge, the network disintegrated into large fragments. A mathematical model predicts the network length as a function of width, actin and ADF/Cofilin concentrations. Local depletion of ADF/Cofilin by binding to actin is significant, leading to wider networks growing longer. A single rate of breaking network nodes, proportional to ADF/Cofilin density and inversely proportional to the square of the actin density, can account for the disassembly dynamics. Selective disassembly of heterogeneous networks by ADF/Cofilin controls steering during motility. Our results establish general principles on how the dynamic steady state of actin network emerges from biochemical and structural feedbacks., Competing Interests: AM, TI, CG, TK, RB, MT, LB, AM No competing interests declared, (© 2019, Manhart et al.)

Gold nanoparticles (AuNPs) and their conjugation to biological samples have numerous potential applications. When combined with cryo-electron microscopy and tomography analysis, AuNPs may provide a versatile and powerful tool to identify and precisely localize proteins even when attached to cellular components. Here, we describe a general and facile approach for the synthesis of homogeneous and stable AuNPs, which can readily be conjugated to a molecule of interest and imaged by cryo-electron tomography (cryo-ET). We demonstrate the synthesis of 2.2 ± 0.45-nm tiopronin-protected AuNPs, followed by their conjugation with recombinant proteins and peptides. Visualization of the ∼2.2-nm gold-tagged peptides by cryo-ET reveals the potential use of this strategy to label and localize accessible proteins in a cellular environment with nanometric resolution., (Copyright © 2018. Published by Elsevier Ltd.)

Recent evidence has suggested that Srv2/CAP (cyclase-associated protein) has two distinct functional roles in regulating actin turnover, with its N-terminus enhancing cofilin-mediated severing of actin filaments and its C-terminus catalyzing actin monomer recycling. However, it has remained unclear to what degree these two activities are coordinated by being linked in one molecule, or whether they can function autonomously. To address this, we physically divided the protein into two separate halves, N-Srv2 and C-Srv2, and asked whether they are able to function in trans both in living cells and in reconstituted assays for F-actin turnover and actin-based motility. Remarkably, in F-actin turnover assays the stimulatory effects of N-Srv2 and C-Srv2 functioning in trans were quantitatively similar to those of intact full-length Srv2. Further, in bead motility assays and in vivo, the fragments again functioned in trans, although not with the full effectiveness of intact Srv2. From these data, we conclude that the functions of the two halves of Srv2/CAP are largely autonomous, although their linkage improves coordination of the two functions in specific settings, possibly explaining why the linkage is conserved across distant plant, animal, and fungal species., (© 2014 Wiley Periodicals, Inc.)

The actin cytoskeleton is a key component of the cellular architecture. However, understanding actin organization and dynamics in vivo is a complex challenge. Reconstitution of actin structures in vitro, in simplified media, allows one to pinpoint the cellular biochemical components and their molecular interactions underlying the architecture and dynamics of the actin network. Previously, little was known about the extent to which geometrical constraints influence the dynamic ultrastructure of these networks. Therefore, in order to study the balance between biochemical and geometrical control of complex actin organization, we used the innovative methodologies of UV and laser patterning to design a wide repertoire of nucleation geometries from which we assembled branched actin networks. Using these methods, we were able to reconstitute complex actin network organizations, closely related to cellular architecture, to precisely direct and control their 3D connections. This methodology mimics the actin networks encountered in cells and can serve in the fabrication of innovative bioinspired systems., (© 2014 Elsevier Inc. All rights reserved.)

The actin cytoskeleton is a fundamental player in many cellular processes. Ultrastructural studies have revealed its extremely complex organization, where actin filaments self-organize into defined and specialized structures of distinct functions and, yet, are able to selectively recruit biochemical regulators that are available in the entire cell volume. To overcome this extraordinary complexity, simplified reconstituted systems significantly improve our understanding of actin dynamics and self-organization. However, little is known regarding physical rules governing actin networks organization and to which extent network structure may direct and regulate selective interactions with specific regulators. Here, we describe the first method to direct actin filament assembly to specific 2D motifs with a finely tuned geometry and relative distribution. This method enables the study of how geometrical confinement governs actin network structural organization and how, in return, structural cues can control selective contraction by myosin motor. The protocol relies on the use of surface micropatterning and functionalization procedures in order to selectively direct actin filament assembly to specific sites of nucleation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cell motility depends on the rapid assembly, aging, severing, and disassembly of actin filaments in spatially distinct zones. How a set of actin regulatory proteins that sustains actin-based force generation during motility work together in space and time remains poorly understood. We present our study of the distribution and dynamics of Arp2/3 complex, capping protein (CP), and actin-depolymerizing factor (ADF)/cofilin in actin "comet tails," using a minimal reconstituted system with nucleation-promoting factor (NPF)-coated beads. The Arp2/3 complex concentrates at nucleation sites near the beads as well as in the first actin shell. CP colocalizes with actin and is homogeneously distributed throughout the comet tail; it serves to constrain the spatial distribution of ATP/ADP-P(i) filament zones to areas near the bead. The association of ADF/cofilin with the actin network is therefore governed by kinetics of actin assembly, actin nucleotide state, and CP binding. A kinetic simulation accurately validates these observations. Following its binding to the actin networks, ADF/cofilin is able to break up the dense actin filament array of a comet tail. Stochastic severing by ADF/cofilin loosens the tight entanglement of actin filaments inside the comet tail and facilitates turnover through the macroscopic release of large portions of the aged actin network.